Bilirubin (B) is a sensitizer of photodynamic damage in the isolated membrane (ghost) of the human red cell (RBC). Included in the damage is intermolecular cross-linking (principally not through -S-S-bonds) of major membrane proteins. A comprehensive study of this process is planned. To learn whether lipid peroxides are involved, we will a) irradiate membrane proteins and B after lipid removal; b) separate cross-linked material from remaining proteins and lipids, and analyze for incorporated phospholipid. We will attempt to identify cross-linked components by extracting various proteins selectively from the membrane prior to insertion of B and irradiation. Since cross-linking might play a role in B-sensitized photohemolysis, we will investigate the occurrence of the former in intact RBC irradiated in the presence of B. Comparative studies with platelets and platelet membranes will be initiated. Our previous work has shown that B photoinactivates glyceraldehyde 3-P dehydrogenase, not only when the enzyme is attached to RBC ghosts, but also after its release. The nature of this effect will be investigated in terms of types of residues destroyed, and their location in the amino acid sequence. The possibility that serum albumin carrying polyunsaturated fatty acids is liable to photodamage as it binds B will also be examined. One manifestation of this, cross-linking as in ghost proteins, will be studied by gel electorphoresis, ultracentrifugation, amino acid analysis, and peptide mapping. Infants with hyperbilirubinemia are often subjected to phototherapy to destroy B and prevent its entry into the central nervous system. A potential hazard of phototherapy, viz. photodamage in cells incorporating B, may become increasingly evident from this work.